Mice
Male WT C57BL/6J mice between 8 and 14 weeks old were used in all experiments and were obtained from Jackson Laboratories. Mice were housed under a 12-hour light/dark cycle with free access to water and standard rodent chow. All animal procedures were approved by and carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of Michigan.
Stereotaxic 6OHDA injection
Mice were anesthetized with 2% isoflurane and secured on a stereotactic frame. 6-hydroxydopamine (6OHDA; 2547; Tocris) was dissolved in a 0.2% ascorbic acid saline solution. WT mice received a unilateral injection of 6OHDA in the striatum. The striatum coordinate used for stereotaxic injection was AP= 0.7 ML=2.0 DV=-2.0 mm. A total of 5 ug of 6OHDA in 2 μL was injected in the striatum at a rate of 0.5 μL/min with a 33G nanofil blunt needle (NF33BL-2, World Precision Instruments) connected to FEP tubbing and a 25uL Hamilton syringe. After 6OHDA infusion, the nanofil blunt needle was left in place for 5 min. 4 weeks after injection, mice were PBS and 4% PFA perfused for immunohistochemistry analysis.
rAAV2-hα-SYN design and production
Recombinant adeno-associated virus serotype 2 (rAAV2) expressing human wild-type α-SYN (rAAV2-hα-SYN) or rAAV2-empty (rAAV2-control) virus were produced at the University of Michigan Vector core. Cloning of hα-SYN was done using the pAAV-hα-SYN WT plasmid as a template (#36055, Addgene) and the pAAV-CBA plasmid as the vector backbone (#81008; Addgene). The resulting plasmid pAAV-CBA-hα-SYN was used to produce rAAV2-hα-SYN virus where hα-SYN expression is driven by the chicken-beta actin (CBA) promoter. The final titers of the viral stocks were determined by qPCR and ranged between 2.0 x 1013 and 3.12 x 1013 viral genome copies per milliliter (vg/mL; plasmids and viruses are available at the University of Michigan Vector core).
Stereotaxic rAAV2-hα-SYN injection in SN
Mice were anesthetized with 2% isoflurane and secured on a stereotactic frame. WT mice received a unilateral injection of rAAV2-hα-SYN at a final concentration of 2 x 1013 vg/mL into the SN. The SN coordinate used for stereotaxic injection was AP=-3.1 ML=-1.4 DV=-4.2 mm. A total of 2 μL of rAAV2-hα-SYN virus was injected in the SN at a rate of 0.25 μL/min with a 33G nanofil blunt needle (NF33BL-2, World Precision Instruments) connected to FEP tubbing and a 25uL Hamilton syringe. After rAAV2-hα-SYN virus infusion, the nanofil blunt needle was left in place for 5 min. 4 weeks after injection, mice were PBS and 4% PFA perfused for immunohistochemistry analysis.
Drug treatments
N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) was prepared in saline solution and injected IP in a single dose of 50 mg/Kg (2958; Tocris). This dose was selected based on previous studies showing tissue NE depletion in the brain (Ross and Stenfors, 2015). 3 days after DSP-4 treatment stereotaxic injection of 6OHDA or rAAV2-hα-SYN injection was performed as described above. To block the norepinephrine transporter (NET) we use Desipramine (D3900; Sigma). A single dose of Desipramine (25 mg/Kg in saline solution) was delivered IP 45 min prior to DSP-4 treatment. Propranolol was delivered IP daily at 10 mg/Kg in saline solution (0624; Tocris). Propranolol treatment started 45 min after stereotaxic injection of rAAV2-hα-SYN injection and was given once a day until the end of the experiment. Xamoterol (3 mg/Kg; IP) (24267; Cayman) and Clenbuterol (10 mg/Kg; IP) (C5423, Sigma) daily treatment was performed 45 min after stereotaxic injection of rAAV2-hα-SYN injection for 4 weeks until the end of the experiment. All drugs used here were dissolved fresh using saline solution the day of treatment and saline solution was used as vehicle control in all experimental conditions. Clenbuterol and xamoterol doses were selected based on previous studies related to CNS pathologies (Mittal et al., 2017; Faizi et al., 2011).
NE and Serotonin measurements
The SN and cortex of WT mice 4 weeks after saline, DSP-4 or Desipramine + DSP-4 treatments were dissected and snap-frozen in liquid nitrogen. Then, samples were sonicated on ice by probe sonication on setting 3 with a 30% duty cycle in ice-cold 0.1M PCA containing 0.1 mM EDTA. Samples were centrifuged at 10,000 × g for 10 minutes at 4ºC. Then, supernatants were transferred into fresh 0.22 µM PVDF microcentrifuge filter tubes. NE and Serotonin concentrations were determined by reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection (HPLC-ED). For HPLC, an ESA 5600A CoulArray detection system, equipped with an ESA Model 584 pump and an ESA 542 refrigerated autosampler was used. Separations were performed at 26ºC using an MD-150 × 3.2 mm C18 column. The mobile phase consisted of 1.6 mM 1-octane sulfonic acid sodium, 75 mM NaH2PO4, 0.025% triethylamine, and 8% acetonitrile at pH 3.0. The samples were eluted isocratically at 0.4 mL/min and detected using a 6210 electrochemical cell (ESA, Bedford, MA) equipped with a 5020-guard cell. Guard cell potential was set at 600 mV, while analytical cell potentials were −175, 150, 350 and 425 mV. The total run time for each run is 40 min. The analytes were identified by the matching criteria of retention time to known standards (Sigma Chemical Co., St. Louis MO). NE and serotonin were quantified by comparing peak areas to those of standards on the dominant sensor. Data were normalized by protein concentration in each brain sample.
Immunohistochemistry
For immunofluorescence, mice were PBS and 4% PFA-perfused, brains were harvested, post-fixed in PFA, dehydrated in 30% sucrose and embedded in OCT. 14μm-thick frozen sections were permeabilized and blocked in 0.5% TritonX-100 and 5% Bovine Serum Albumin in 0.5% TritonX-100/1X PBS, respectively. Sections were incubated with primary antibodies in blocking solution overnight at 4°C followed by incubation with 488, 564 and 647 Alexa-Fluor conjugated secondary antibodies for 1h at room temperature. After secondary antibody incubations sections were washed with 0.1% NP-40/1X PBS. DAPI was used to detect cells nuclei. Sections were mounted using ProLong™ Diamond Antifade Mountant (P36961; Life Technologies). The primary antibodies used were: Tyrosine Hydroxylase (TH) (1:1000; ab113, Abcam), hα-SYN (1:300; ab138501, Abcam), TMEM119 (1:500; ab209064, Abcam), CD16/32 (1:100; #101301, BioLegend) and CD3 (1:200; 100347, BioLegend). For staining involving hα-SYN, heat-mediated antigen retrieval was performed using DAKO retrieval solution (S1700; DAKO). All images were acquired with a Nikon Ti-E Eclipse Microscope using a 20x objective (Plan-Apo, 0.75 numerical aperture) or a 60x oil objective (Plan-Apo, 1.4 numerical aperture). Confocal images were acquired using CREST X-Light V2 Spinning Disk. Z-stacks of 60x images were collected at 0.5 μm increments with a total thickness of ~6 μm. Images were captured with an ORCA-fusion camera (C14440-20UP, Hamamatsu) or an Andor Zyla camera (Zyla 4.2 sCMOS, Oxford Instruments). Large scan function with Z step focus was used to capture stitched images of the SN using a 20x objective. Confocal images are shown as maximum intensity projection images. Images are representative of the respective staining and were processed and analyzed using NIS-elements advanced research software (Nikon Instrument Inc) and FIJI-Image J open software.
Quantification of dopaminergic neurons
Estimation of dopaminergic neuron degeneration in the SN was performed by counting the total number of TH+ neurons in the injected and uninjected SN 4 weeks after rAAV2-hα-SYN. 14µm-thick coronal brain sections were sampled at intervals of 112 µm through the rostrocaudal extent of the SN, at least 4 sections per mouse were counted. The total number of TH+ neurons (Nt) in the injected and uninjected hemisphere of the SN were estimated using model-based stereology incorporating the Abercrombie correction with the following formula: Nt = Ns × (St/Ss) × M/(M + D), where Ns is the total number of neuron counted, St is the total number of sections in the brain region, Ss is the number of sections sampled, M is the thickness of the section, and D is the average diameter of the counted neurons [20]. The average diameter of TH neurons was calculated averaging the min and max Feret diameter. Nt for the uninjected SN was used as an internal control in every mouse and a % of dopaminergic neuron survival was obtained using the following formula: TH+ neuron survival (%) = (Nt injected SN/ Nt uninjected SN) x 100. The genotype of mice was unknown to the investigator at the time of quantification.
Fluorescence intensity quantification
Fluorescence intensity of TMEM119 and CD16/32 in the injected and uninjected SN 4 weeks after rAAV2-hα-SYN virus injection was performed as follows: 14µm-thick coronal brain sections co-stained with TH, TMEM119 and CD16/32 were sampled on intervals of 112 µm through the rostrocaudal extent of the SN, 4 sections per mouse were quantified. A region of interest defining the entire SN pars compacta (SNpc) using TH co-staining was used to quantify the sum fluorescence intensity above background in the injected and uninjected SN. A relative fluorescence value was calculated in each mouse using the uninjected SN as an internal control. For the average number of CD3+ cells quantification in the SN 4 weeks after rAAV2-hα-SYN injection, 14µm-thick coronal brain sections were stained with CD3 and TH. The average number of CD3+ cells associated with the SNpc were counted by defining the SNpc using TH staining and the average number of CD3+ cells were then estimated in the injected and uninjected (rarely observed) SNpc in 4 sections per mouse sampling the rostrocaudal extent of the SN.
Corridor task
Lateralized sensory-motor integration was measured using a corridor task. This behavioral test was selected due to its sensitivity to detect partial unilateral dopaminergic neuron damage in rats and mouse PD models [21, 22], which is ideal for the mild unilateral degeneration of dopaminergic neurons observed in the SN after rAAV2-hα-SYN injection. This test consists in a long narrow rectangular plexiglass corridor with the following dimensions: L=60 cm x W=4 cm x H=15 cm (testing corridor). The testing corridor contains 10 pairs of adjacent Eppendorf caps placed at 5-cm intervals with 4-5 sugar pellets each (20 mg; TestDiet). A corridor without Eppendorf caps with the same dimensions as the testing corridor was used as the habituation corridor. WT mice treated with clenbuterol or saline daily after rAAV2-hα-SYN virus injection were habituated 1 day before the end of the experiment (4 weeks after injection) in the habituation corridor by scattering sugar pellets along the corridor floor and allowing them to freely explore for 10 min. Lateralized sensory-motor integration was tested 4 weeks after rAAV2-hα-SYN injection. On the testing day, mice were placed in the habituation corridor for 5 min in the absence of sugar pellets, then mice were transferred to one end of the testing corridor containing sugar pellets and video recorded for 5 min. The video recordings were analyzed by an investigator blinded to the treatment and genotype. The number of ipsilateral and contralateral explorations relative to the injected hemisphere were counted until the mouse made a total of 20 explorations or the video ended. An exploration was defined as a nose-poke into an Eppendorf cap, whether the sugar pellet was poked or eaten, and a new exploration was only counted by exploring a new cap. Data is expressed as a percentage of bias lateralized explorations, calculated as: Bias (%) = 100*(ipsilateral - contralateral) / (ipsilateral+contralateral).
Statistics
Data analysis was performed using GraphPad Prism 8 statistical software (GraphPad Software, La Jolla, CA, USA). All experiments were repeated at least two independent times and n indicates the number of individual mice used in the study. For statistical analysis, in any experiment with only two groups, a two-tailed t-test was used. For experiments with more than two groups, a one-way ANOVA with Tukey or Dunnet post hoc test was used. Data is represented as mean values ± S.E.M; p <0.05 was considered significant.